In liquid chromatography, samples are separated by a chromatographic column. The components separated from the sample are traditionally detected by means of a refractometer or a spectrophotometer. Since the refractometer does not distinguish one chemical from another, a spectrophotometer, in particular a UV spectrophotometer, is preferred. Double beam U.V. spectrophotometers have been employed in liquid chromatography, particularly high pressure liquid chromatography (HPLC) for many years.
One such double beam U.V. spectrophotometer for HPLC was described in Schoeffel et al, U.S. Pat. No. 3,985,441. The optical system employed a pair of multidirectional refocusing optical mirrors to direct two identical areas of light from a single light source onto a single grating which disperses the beams into a pair of spectra. A pair of apertures permit only light with a very narrow selected range of wavelengths from the pair of spectra to be passed through a pair of optical cells. The emergent light beams impinge on a pair of photodetectors, the signals of which are converted to absorbance units by an analog logarithmic ratio circuit.
However, conventional UV spectrophotometers used for HPLC can only record absorbance at a single narrow band of wavelengths. Thus, not all of the sample components can be detected at their optimum absorbance. In fact, some components may not be detected at all, if these components did not absorb UV light at the selected wavelengths.
Attempts have been made to develop multiwavelength spectrophotometers for HPLC. The first generation of such instruments employed moving mechanical parts to scan the spectrum, typified by using rotating mirrors or vibrating galvanometers. The second generation utilized Vidicon tubes. However, these systems are very expensive and tend to show memory and blooming which makes them suitable as interim solutions only. See Dessy et al., J. of Chroma. Sci., 14, pp. 195-201 (April 1976).
Dessy et al. describes one system which employed photodiode arrays to provide a means for recording the spectrum of each component separated in HPLC. The light from the source, a deuterium lamp or a xenon lamp, is directed by a light pipe of optical fibers onto the sample and reference cells of a HPLC unit. The light is then redirected by means of light pipes to a pair of concave holographic gratings to focus and disperse the light beams onto a pair of photodiode arrays. The signals from the photodiode array are processed through an analog-log converter circuit into readable form. The total amount of time required to record a spectrum is about 3.1 seconds. This is much too slow to make recordings of the spectra of components as these are being eluted from the chromatographic system. Moreover, the geometry of the optical systems is such that the photodiode array must be offset away from the flow cells to avoid interference from the undispersed light beam. This means that the housing for the optical systems is rather bulky.
Another early dual beam spectrophotometer was commercialized by Hitachi. The Hitachi 635M dual beam channel UV detector utilized deuterium lamp as the source. The beams were split, directed through a pair of flow cells on to an astigmatic concave replica grating which dispersed and at the same time focused the beams onto photocell arrays with eight channels. The signals from the eight pairs of photocell array with one pair for each selected wavelength were monitored. This represented an improvement over single wavelength recording. However, it can not be utilized for full spectrum recording. Moreover, it has a similar optical arrangement to Dessy et al. and has similar disadvantages.
U.S. Pat. No. 4,678,917 issued July 7, 1987, describes a method and apparatus for taking instantaneous readings from a multichannel spectrophotometer. The beam from a light source is split and both beams were respectively directed to reflectors and passed through a sample cell and a reference cell. The radiation from each of the cells was directed to a diffraction grating which disperse the beam into a polychromatic divergent beam and direct the beam on a linear array of photodetectors. The signals from the photodetectors were carried through separate signal channels into sample-and-hold circuits controlled by a multiplex switch which operates in response to control signals from a sequence control. The multiplex switch causes the sample-and-hold circuits to sample and hold the signals which are read out of the analog mode and converted to digital mode. The digitized data is stored and processed by a data processing unit which may be a digital computer and presented as absorbance units versus wavelength. This instrument is capable of full spectrum recording.
The spectrophotometer described in U.S. Pat. No. 4,678,917 has several disadvantages. The analog-to-digital converter utilized in U.S. Pat. No. 4,678,917 calls for complex circuitry which is rather costly. The optical system as depicted produces a pair of unfocused divergent beams on the photodiode arrays and will cause problems in increased noise level. Moreover, the heat from the light source being close to the sample cells, would affect the cells and cause additional errors in the results obtained. Thus to avoid all of these problems, a different optical arrangement is required. It is the object of the present invention to develop a dual beam spectrophotometer that is accurate, compact and stable, but less costly.
It is another objective of the present invention to develop a dual beam spectrophotometer with a simple and less costly optical system wherein the light source, the reference and sample cells and the photodetectors are isolated from one another resulting in a system which is a stable and has very low drift.
It is a further objective of the present invention to develop a signal converter for the simultaneous recording of the signals from a multichannel full spectrum spectrophotometer that utilizes a simple circuitry and is less costly.